LOCUS       sequence-171151-        7602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express I-PhoCl-HCVp with N-term EBFP2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7602)
  AUTHORS   Zhang W, Lohman AW, Zhuravlova Y, Lu X, Wiens MD, Hoi H, Yaganoglu 
            S, Mohr MA, Kitova EN, Klassen JS, Pantazis P, Thompson RJ, Campbell
            RE
  TITLE     Optogenetic control with a photocleavable protein, PhoCl.
  JOURNAL   Nat Methods. 2017 Mar 13. doi: 10.1038/nmeth.4222.
  PUBMED    28288123
REFERENCE   2  (bases 1 to 7602)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2017 Mar 13. doi: 10.1038/nmeth.4222."
COMMENT     Sequence Label: pcDNA-I-PhoCl-HCVp
FEATURES             Location/Qualifiers
     source          1..7602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      898..907
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(916..918,919..921,922..1632)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(961..982)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             1801..2526
                     /codon_start=1
                     /product="photocleavable protein that separates into two 
                     fragments upon exposure to violet light (Zhang et al., 
                     2017)"
                     /label=PhoCl
                     /translation="VIPDYFKQSFPEGYSWERSMTYEDGGICIATNDITMEGDSFINKI
                     HFKGTNFPPNGPVMQKRTVGWEASTEKMYERDGVLKGDVKMKLLLKGGGHYRCDYRTTY
                     KVKQKPVKLPDYHFVDHRIEILSHDKDYNKVKLYEHAVARNSTDSMDELYKGGSGGMVS
                     KGEETITSVIKPDMKNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDLEVKEGAPLPFA
                     YDILTTAFHYGNRVFTKYPR"
     primer_bind     complement(3196..3213)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3202..3426
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3472..3900
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3559..3578)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3769..3790
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3909..3929)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3914..4243
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4094..4229
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4156..4175
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4310..5104
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4364..4383)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4974..4993
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5278..5399
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5315..5334)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5369..5388
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5448..5464)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5448..5464)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5461..5483)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5472..5488
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5496..5502,5503..5520,5521..5526))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5541..5562
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5679..5696)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5850..6435)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5930..5949)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6606..7397,7398..7466))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7229..7248
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7467..7571)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctagccgcc
      901 accatggcct cgagtatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      961 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgagggg cgagggcgag
     1021 ggcgatgcca ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1081 gtgccctggc ccaccctcgt gaccaccctg agccacggcg tgcagtgctt cgcccgctac
     1141 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1201 gagcgcacca tcttcttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc
     1261 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggcg tcgacttcaa ggaggacggc
     1321 aacatcctgg ggcacaagct ggagtacaac ttcaacagcc acaacatcta tatcatggcc
     1381 gtcaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacgt ggaggacggc
     1441 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1501 ctgcccgaca gccactacct gagcacccag tccgtgctga gcaaagaccc caacgagaag
     1561 cgcgatcaca tggtcctgct ggagttccgc accgccgccg ggatcactct cggcatggac
     1621 gagctgtaca aggaattcag cgggtcaggc ggatctggaa gtggtggtgg agaacttgat
     1681 gaattggtat acttactaga tgggccaggt tatgacccta tacatagtgg cggtagtggg
     1741 ggatctggtg gctcaggagg gagcggatcc atggtgtcca agggcgaaga gacaaccatg
     1801 gtgatccctg actacttcaa gcagagcttc cccgagggct acagctggga gcgcagcatg
     1861 acctacgagg acggcggcat ctgcatcgcc accaacgaca tcacaatgga gggggacagc
     1921 ttcatcaaca agatccactt caagggcacg aacttccccc ccaacggccc cgtgatgcag
     1981 aagaggaccg tgggctggga ggccagcacc gagaagatgt acgagcgcga cggcgtgctg
     2041 aagggcgacg tgaagatgaa gctgctgctg aagggcggcg gccactatcg ctgcgactac
     2101 cgcaccacct acaaggtcaa gcagaagccc gtaaagctgc ccgactacca cttcgtggac
     2161 caccgcatcg agatcctgag ccacgacaag gactacaaca aggtgaagct gtacgagcac
     2221 gccgtggccc gcaactccac cgacagcatg gacgagctgt acaagggtgg cagcggtggc
     2281 atggtgagca agggcgagga gaccattaca agcgtgatca agcctgacat gaagaacaag
     2341 ctgcgcatgg agggcaacgt gaacggccac gccttcgtga tcgagggcga gggcagcggc
     2401 aagcccttcg agggcatcca gacgattgat ttggaggtga aggagggcgc cccgctgccc
     2461 ttcgcctacg acatcctgac caccgccttc cactacggca accgcgtgtt caccaagtac
     2521 ccacggggag gtagcaccga cggcatggac gagctgtaca agtctagagg cagtggaaca
     2581 gggggttctg ttgttattgt tggtagaatt attttatctg gtagtggtag tatcacggcc
     2641 tactcccaac agacgcgggg cctacttggt tgcatcatca ctagcctcac aggccgggac
     2701 aagaaccagg tcgaagggga ggttcaagtg gtttctaccg caacacaatc tttcctggcg
     2761 acctgcgtca acggcgtgtg ctggactgtc taccatggcg ctggctcgaa gaccctagcc
     2821 ggtccaaaag gtccaatcac ccaaatgtac accaatgtag accaggacct cgtcggctgg
     2881 caggcgcctc caggggcgcg ctccttgaca ccatgcacct gtggcagctc ggacctttac
     2941 ttggtcacga gacatgctga tgtcattccg gtgcgccggc gaggcgacag caggggaagt
     3001 ctactctccc ccaggcccgt ctcctacctg aaaggctcct cgggtggtcc attgctttgc
     3061 ccttcggggc acgctgtggg catcttccgg gctgctgtgt gcacccgggg ggtcgcgaag
     3121 gcggtggact tcgtgcccgt tgagtctatg gaaactacca tgcggtctta aaagcttggt
     3181 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3241 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3301 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3361 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     3421 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3481 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3541 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3601 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3661 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     3721 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3781 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3841 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3901 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3961 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     4021 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     4081 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4141 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     4201 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     4261 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4321 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4381 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4441 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     4501 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4561 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4621 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4681 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4741 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4801 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     4861 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4921 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4981 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     5041 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     5101 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     5161 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     5221 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     5281 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     5341 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     5401 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     5461 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5521 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5581 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5641 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5701 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5761 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5821 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5881 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5941 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6001 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6061 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6121 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6181 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6241 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6301 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6361 ggcaaacaaa ccaccgctgg tagcggtttt tttgtttgca agcagcagat tacgcgcaga
     6421 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6481 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6541 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6601 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6661 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6721 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6781 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6841 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6901 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6961 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     7021 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     7081 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7141 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7201 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7261 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7321 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7381 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7441 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7501 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7561 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
